The CBP and p300 paralogs were identified by their roles in regulating cyclic AMP-related gene activation and binding to adenoviral protein E1A, respectively (Chrivia, et al. (1993) Nature 365:855-859; Eckner, et al. (1994) Genes Dev. 8:869-884). One or more copies of the p300/CBP transcriptional coactivator is encoded in organisms from worm to man, and they have been intensively studied because of their diverse and important roles in complex biological processes (Goodman & Smolik (2000) Genes Dev. 14:1553-1577). The p300/CBP protein is ˜250 kDa and contains a number of well-defined domains, many of which are crucial for its recruitment by a wide range of transcription factors (Legge, et al. (2004) J. Mol. Biol. 343:1081-1093; Mujtaba, et al. (2004) Mol. Cell. 13:251-263; Radhakrishnan, et al. (1997) Cell 91:741-752). p300/CBP has been shown to possess intrinsic histone acetyltransferase (HAT) activity which has led to a wide array of insights into its biological activities (Bannister & Kouzarides (1996) Nature 384:641-643; Ogryzko, et al. (1996) Cell 87:953-959). For example, p300/CBP HAT activity is important for its ability to act as a coactivator for a variety of transcription factors, e.g., p53, NFκB, STAT3, GATA-1, MyoD, TCF, androgen receptor (AR), and HIV Tat; thereby indicating that its HAT activity is important for a variety of pathways including cancer (Iyer, et al. (2004b) Oncogene 23:4225-4231), HIV (Kaehlcke, et al. (2003) Mol. Cell. 12:167-176) and HTLV-1 (Georges, et al. (2003) Mol. Cell. Biol. 23:3392-3404) pathogenesis; as well as, cardiac remodeling (Gusterson, et al. (2003) J. Biol. Chem. 278:6838-6847), glucose regulation (van der Heide & Smidt (2005) Trends Biochem. Sci. 30:81-86), oxygen sensing (Roe, et al. (2006) Mol. Cell 22:395-405), and steroid hormone signaling (Korzus, et al. (1998) Science 279:703-707). In addition to catalyzing the acetylation of multiple lysines on all four core histones, p300/CBP has been shown to acetylate a wide array of transcription factors and other proteins as part of its functions. Some of these p300/CBP-acetylated substrates include p53, p73, NFκB, STAT3, GATA-1, MyoD, TCF, E2F1, HMG14, HMGI(Y), androgen receptor (AR), Tat, and c-Myb (Chen, et al. (2001) Science 293:1653-1657; Costanzo, et al. (2002) Mol. Cell. 9:175-186; Thompson, et al. (2001) J. Biol. Chem. 276:33721-33729; Yuan, et al. (2005) Science 307:269-273). Although there are no precise consensus sequences for p300/CBP-mediated acetylation, there is a clear preference for nearby positively charged residues influencing targeted lysine acetylation (Thompson, et al. (2001) supra).
As noted above, p300/CBP plays a key role in regulating the transcription of a large subset of eukaryotic genes. Consistent with this important role is the fact that mutations, altered expression, and gene rearrangements of p300/CBP have been observed in a variety of diseases including cancer (Iyer, et al. (2004b) supra). For example, patients with Rubinstein-Taybi Syndrome, which involves a heterozygous mutation in one CBP allele (Murata, et al. (2001) Hum. Mol. Genet. 10:1071-1076), have an increased incidence of tumors. Additionally, point mutations thought to interfere with the catalytic activity of p300/CBP have been observed in pancreatic cancer (Gayther, et al. (2000) Nat. Genet. 24:300-303), colon cancer (Muraoka, et al. (1996) Oncogene 12:1565-1569), and lung cancer (Kishimoto, et al. (2005) Clin. Cancer Res. 11:512-519). Gene fusion events involving the CBP HAT domain have been detected in a number of acute leukemias (Borrow, et al. (1996) Nat. Genet. 14:33-41). Finally, overexpression of p300/CBP has been detected in a variety of cancers including colon (Pena, et al. (2006) Int. J. Cancer 119:2098-2104), gastric (Kim, et al. (2007) Am. J. Physiol. Cell Physiol. 292:C857-866), and thyroid (Fluge, et al. (2006) Thyroid 16:161-175) carcinoma.
The coactivator activity of p300/CBP is controlled at multiple levels and its regulation has been studied (Goodman & Smolik (2000) supra). For example, p300/CBP is known to be phosphorylated, methylated, ubiquitinated, sumoylated, and acetylated and these modifications exert a myriad of effects on p300/CBP coactivator activity by modulating its protein levels, its interactions with other proteins, and its HAT activity (Goodman & Smolik (2000) supra; Thompson, et al. (2004) Nat. Struct. Mol. Biol. 11:308-315). Regarding acetylation, there are a dense cluster of lysines in a flexible loop region of the p300/CBP HAT domain that are sites of autoacetylation (Thompson, et al. (2004) supra). Intermolecular autoacetylation of these lysines appears to upregulate p300/CBP HAT activity (Thompson, et al. (2004) supra) and also modulate protein-protein interactions with APC (Turnell, et al. (2005) Nature 438:690-695), PIC (Black, et al. (2006) Mol. Cell. 23:809-818), and ATF-2 among possibly others. Partial deletion of this p300/CBP autoacetylated loop can upregulate HAT activity and modulate transcriptional activation (Thompson, et al. (2004) supra).
Studies investigating the structure, mechanism, and inhibition of different HATs have been conducted. The most well-understood HATs are the paralogs PCAF and GCN5, and these enzymes appear to be classical members of the GNAT superfamily (Neuwald & Landsman (1997) Trends Biochem. Sci. 22:154-155) based on structure and catalytic mechanism (Vetting, et al. (2005) Arch. Biochem. Biophys. 433:212-226). The GNAT superfamily is composed of weakly conserved acetyltransferases with ˜200 residue catalytic domains that show a similar core protein fold and include enzymes involved in antibiotic resistance, melatonin biosynthesis, and polyamine metabolism (Vetting, et al. (2005) supra). The catalytic mechanism of PCAF/GCN5 and most other GNATs usually involves a ternary complex mechanism with ordered binding of the acetyl-CoA substrate prior to the amine substrate (Vetting, et al. (2005) supra). Upon ternary complex formation, there is direct transfer of the acetyl group from acetyl-CoA to the substrate amino group. The α-β fold for acetyl-CoA binding is quite conserved and many of these enzymes appear to have a catalytic base assisting in amine substrate deprotonation (Vetting, et al. (2005) supra). Bisubstrate analog inhibitors in which the amine substrate are linked to coenzyme A via an acetyl bridge have proved to be powerful inhibitors for these enzymes and have been extensively used in biochemical and structural studies (Vetting, et al. (2005) supra).
Sequence alignments and enzymology experiments on p300/CBP have led to somewhat confusing and contradictory results regarding the mechanism of p300/CBP and its structural relationship to PCAF/GCN5. For example, sequence alignments of p300/CBP and PCAF/GCN5 (Martinez-Balbas, et al. (1998) EMBO J. 17:2886-2893; Yuan & Giordano (2002) Oncogene 21:2253-2260) have shown limited homology that appears to be inconsistent with the PCAF/GCN5 structure (Poux, et al. (2002) Proc. Natl. Acad. Sci. USA 99:14065-14070). Additionally, a two substrate kinetic analysis showed a parallel line pattern suggestive of a ping-pong kinetic mechanism with a covalent enzyme intermediate (Thompson, et al. (2001) supra), potentially similar to the mechanism employed by Esa1 (Berndsen, et al. (2007) Biochemistry 46:623-629) and different from PCAF/Gcn5 (Tanner, et al. (1999) J. Biol. Chem. 274:18157-18160; Trievel, et al. (1999) Proc. Natl. Acad. Sci. USA 96:8931-8936; Vetting, et al. (2005) supra). However, experiments with acetyl-CoA-based affinity labeling agents failed to identify a key active site nucleophilic residue that would play a role in forming a covalent intermediate. Interestingly, the nominal bisubstrate analog Lys-CoA in which a derivatized lysine is bridged to coenzyme A via an acetyl linker is a powerful and selective inhibitor of p300/CBP (Lau, et al. (2000) Mol. Cell. 5:589-595). Paradoxically, longer peptide-CoA conjugates, based on better peptide substrates of p300 HAT, are weaker p300 HAT inhibitors (Lau, et al. (2000) supra). This pattern is reversed for PCAF/GCN5 where longer rather than shorter peptide-CoA conjugates are better HAT PCA Compound F/GCN5 inhibitors (Lau, et al. (2000) supra), consistent with their substrate behaviors and ternary complex mechanisms. Interestingly, deletion of the 3′-phosphate from Lys-CoA results in a 30-fold reduction in p300 HAT inhibitory potency (Cebrat, et al. (2003) Bioorg. Med. Chem. 11:3307-3313). In contrast, for a GNAT superfamily member serotonin N-acetyltransferase bisubstrate analog the 3′-phosphate is essentially completely dispensable (Khalil, et al. (1999) Proc. Natl. Acad. Sci. USA 96:12418-12423). Taken together, these studies provide little information of the nature of p300/CBP HAT mechanism and structure.